Project Summary The insulin/IGF-like signaling (IIS) pathway is highly conserved among animals and is essential for proper metabolism, development, and reproduction. However, IIS activity is also paradoxically variable, resulting in species-specific differences, including body size and fecundity. In humans, IIS heterogeneity may also predispose individuals to certain diseases and disorders. Therefore, an understanding of the broad consequences of differential IIS and its underlying genetic mechanisms will provide insight into development, oogenesis, and disease susceptibility. In Drosophila, IIS is highly divergent between species and, in D. melanogaster and D. sechellia, has been shown to influence fecundity by affecting the number of ovarioles. Utilizing the enormous genetic toolset in the Drosophila ovary, I aim to determine the significance of differential IIS as it pertains to ovariole development in D. melanogaster and D. sechellia. In Aim 1, I will determine the biological significance of differential IIS in D. melanogaster and D. sechellia at the organismal level. By examining life history traits including lifespan, length of development, and stress resistance, I will obtain a comprehensive understanding of how differential IIS broadly affects these species. Moreover, I will measure the active components of the IIS pathway to identify the causal gene(s) that mediate differential IIS. Aim 2 details a plan to find the genetic basis underlying differential IIS between D. melanogaster and D. sechellia by using a multispecies comparative analysis approach to identify regulatory regions that correlate with IIS activity. Coding regions and cis-regulatory regions of candidate genes from D. melanogaster and D. sechellia will be tested for their activity. In Aim 3, I will perform a genome-wide RNAi screen to find IIS-dependent factors that influence ovariole development. The RNAi constructs will encompass components of all known signaling pathways in Drosophila as well as Sirtuins and a subset of meiotic proteins whose mutants show phenotypes similar to those in the IIS pathway. These studies will contribute towards the identification of the genetic mechanisms and IIS-dependent pathways that mediate ovariole formation, fecundity, and overall health.